Use of glycerol-containing media to study the intrinsic fluorescence properties of detergent-solubilized native or expressed SERCA1a.
Rapid irreversible inactivation of Ca (2+)-free states of detergent-solubilized SERCA1a (sarco-endoplasmic reticulum calcium ATPase 1a) has so far prevented the use of Trp fluorescence for functional characterization of this ATPase after its solubilization in various detergents. Here we show that using 20-40% glycerol for protection makes this fluorescence characterization possible. Most of the ligand-induced Trp fluorescence changes previously demonstrated to occur for SERCA1a embedded in native sarcoplasmic reticulum membranes were observed in the combined presence of glycerol and detergent, although the results greatly depended on the detergent used, namely, octaethylene glycol mono- n-dodecyl ether (C 12E 8) or dodecyl maltoside (DDM). In particular, at pH 6, we found a C 12E 8-dependent unexpectedly huge reduction in SERCA1a affinity for Ca (2+). We suggest that a major reason for the different effects of the two detergents is that high concentrations of C 12E 8, but not of DDM, slow down the E2 to E1 transition in solubilized and delipidated SERCA1a. Independently of the characterization of the specific effects of various detergents on SR vesicles, our results open the way to functional characterization by Trp fluorescence of heterologously expressed and purified mutants of SERCA1a in the presence of detergent, without their preliminary reconstitution into liposomes. As an example, we used the E309Q mutant to demonstrate our previous suspicion that Ca (2+) binding to Site I of SERCA1a in fact slightly reduces Trp fluorescence, and consequently that the rise in this fluorescence generally observed when two Ca (2+) ions bind to WT SERCA1a mainly reflects Ca (2+) binding at Site II of SERCA1a.
Montigny
Cédric
C
CNRS, URA 2096, Systemes membranaires, photobiologie, stress et detoxication, F-91191 Gif-sur-Yvette, France.
Arnou
Bertrand
B
Marchal
Estelle
E
Champeil
Philippe
P
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry*; D015870:Gene Expression; D005960:Glucosides / Q000737:chemistry*; D005990:Glycerol / Q000737:chemistry*; D006801:Humans; D020125:Mutation, Missense; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013050:Spectrometry, Fluorescence / Q000379:methods
D003902:Detergents; D005960:Glucosides; D011994:Recombinant Proteins; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; C040358:dodecyl maltoside; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506943:ATP2A1 protein, human; D005990:Glycerol; D002118:Calcium